The invention relates to the production of mammals with defined genetic properties, particularly the production of transgenic animals.
Transgenic animals are organisms into whose germline permanent genetic changes have been introduced; a newly introduced gene is known as a transgene. Transgenic animals constitute an essential tool in modern biology for analysing the tissue-specific regulation of genes and their function in development and in diseases. Moreover, transgenic technology provides an opportunity of having animal models available for diseases in humans and producing large amounts of proteins in farm animals.
In the method of producing transgenic animals which has hitherto been used most frequently, recombinant DNA is microinjected into the fertilized eggs; another technique for introducing genes into animal embryos makes use of viruses, usually recombinant retroviral vectors (cf. the summarising articles by Wagner and Keller, 1992).
The third and most recent technique for introducing foreign genetic material into animals makes use of the potential of embryonic stem cells (ES cells) to create chimeric animals. Mammalian embryos have the capacity to incorporate foreign cells during their development. Two different pre-implantation embryos, usually morulae, are aggregated in vitro; this produces a chimeric embryo, which constitutes a mixture of the two embryos. These embryos are then transferred into a pseudo-pregnant mouse which acts as a foster mother; the chimeric offspring obtained have, in their tissues, different numbers of cells which originate from one of the two original embryos. Combining this method with the use of ES cells has proved very effective in the production of genetically manipulated animals.
Embryonic stem cells are derived from the inner cell mass (ICM) of blastocysts; they are totipotent cells which are capable of developing into all cell lineages, including germ cells, when introduced into an embryo by injection into diploid blastocysts or by aggregation with morulae (Robertson, 1987; Bradley, 1987; Beddington and Robertson, 1989; Nagy et al., 1990). ES cells can be isolated from blastocysts and then established as permanent cell lines if they are cultivated under well defined culture conditions which are strictly adhered to; they can be genetically manipulated. In view of this ability, they constitute an effective tool for modifying the mammalian and particularly the mouse genome by being introduced into the animals, for example, by means of controlled mutations or other genetic modifications (Wagner et al., 1991; Ramirez-Solis et al., 1993; Skarnes, 1993; Bronson and Smithies, 1994).
For some time, cells designated xe2x80x9cembryonic germ cellsxe2x80x9d (EG cells) have been available, which can be cultivated from primordial germ cells into immortalised cell lines and are similar to ES cells in many respects; EG cells are, inter alia, totipotent, can be manipulated in the same way as ES cells and form germline chimeras when introduced into blastocysts (Donovan et al., 1997).
In recent years, various experimental techniques have been developed for producing animals derived from totipotent cells. (Totipotent cells are cells with the ability to differentiate themselves into all somatic cells as well as germ cells.) In the case of ES cells the primary objective of these methods was to obtain the entire developmental potential of ES cells in vitro (Williams et al., 1988; Smith et al., .1988) and to restrict the developmental potential of the host cells in the formation of chimeras and thus increase the frequency of forming germline chimeras (Nagy et al., 1990; Kaufman and Webb, 1990). One of the most important pieces of progress in the development of these techniques is the use of tetraploid embryos as host cells, because tetraploid cells have only restricted potential for development after they have been implanted (Nagy et al., 1990; Kaufmann and Webb, 1990; Kubiak and Tarkowski, 1985). When tetraploid embryos are aggregated with diploid embryos, the differentiation of the tetraploid cells is largely restricted to the primitive endoderm and the trophectoderm, which subsequently form extraembryonic tissue, whereas the diploid cells can form the actual embryo (James and West, 1994; James et al., 1995).
In an earlier study, various ES cell lines were aggregated with morulae in order to produce fetuses which are derived completely from ES cells (organisms derived completely from ES cells are hereinafter referred to as ES animals, e.g. ES mice or ES fetuses; however, the ES foetuses obtained died at birth (Nagy, 1990). Further studies showed that viable, fertile ES mice derived exclusively from ES cells can be obtained if wild-type R1 cells of an earlier passage (Nagy et al., 1993) or TT2 cell lines (Ueda et al., 1995) are used for the aggregation with tetraploid morulae.
Moreover, ES mice are produced by injecting ES cells into diploid blastocysts in a first step, thereby initially obtaining chimeric mice; further crosses produced ES mice after two generations. The method of injecting into blastocysts was first described by Gardner, 1968, and a simplified version was described by Bradley and Robertson, 1986, and by Bradley, 1987.
The objective of obtaining viable ES mice by using ES cells from later passages has not been achieved with the methods available hitherto (Nagy et al., 1993); it did not seem possible to produce ES mice at all using genetically modified ES cells. (The possibility of using ES cells from later passages is significant particularly in view of the use of cell lines and also with respect to the use of genetically modified ES cells the selection of which naturally goes hand in hand with an increase in the number of passages.)
The aim of the present invention was to provide a new process by which mammals with defined genetic properties, particularly transgenic mammals, can be obtained which are derived completely from totipotent cells.
The objective is achieved by means of a process for producing mammals with defined genetic properties, particularly transgenic mammals, wherein totipotent cells of the same mammalian species are introduced into blastocysts and the resulting embryo is transferred into a foster mother. The process is characterised in that totipotent cells with defined genetic properties are introduced into tetraploid blastocysts.
Using the process according to the invention it is possible to obtain animals which are completely derived from totipotent cells. The process according to the invention has the advantage that animals derived totally from totipotent cells are obtained in a single step from totipotent cells cultivated in vitro (ES cells or EG cells).
The term xe2x80x9ctransgenic mammalsxe2x80x9d includes, for the purposes of the present invention, animals which have a permanent genetic modification of any kind.
Animals which xe2x80x9care totally derived from totipotent cellsxe2x80x9d preferably contain up to 100% of cells originating from ES cells or EG cells. However, the animals may contain a small proportion, preferably not more than 10%, of cells derived from the tetraploid blastocysts.
In a preferred embodiment of the invention the mammals are mice; however, the process may also theoretically be applied to all mammals from which ES cells or EG cells can be obtained. The prerequisite for obtaining totipotent cells from mammals other than mice is the definition of conditions which allow the cultivation of ES cells or primordial germ cells from these organisms and the establishing of ES or EG cell lines, which include, inter alia, the need for specific growth factors as well as feeder cells for co-cultivation with the ES cells or EG cells. These conditions can be determined empirically by series of tests.
The isolation of ES cells from blastocysts, the establishing of ES cell lines and their subsequent cultivation are carried out by conventional methods as described, for example, by Doetchmann et al., 1985; Li et al., 1992; Robertson, 1987; Bradley, 1987; Wurst and Joyner, 1993; Hogen et al., 1994; Wang et al., 1992. The cultivation of EG cells can be carried out using methods described in the summarising article by Donovan et al., 1997, and in the original literature cited therein. Totipotent cell lines, e.g. mouse ES cell lines, can be tested in preliminary trials to see whether they are suitable for use in the present invention on the basis of their development potential. To find this out, cells of the lines in question may be injected into diploid mouse embryos, the resulting embryos are introduced into foster mothers and the young are examined for their chimerism rate and for the frequency of formation of germline chimeras.
In a preferred embodiment of the invention the totipotent cells are ES cells.
Tetraploid blastocysts may be obtained by known methods by electrofusion of two-cell embryos and subsequently cultured as described, for example, by James et al., 1992; Nagy and Rossant, 1993; or by Kubiak and Tarkowski, 1985.
The introduction of the ES cells or EG cells into the blastocysts is also carried out in a manner known per se. The preferred method for the purposes of the present invention is the microinjection method as described by Wang et al., 1991, for example. In conventional microinjection, about 5-10 ES cells taken from a single cell suspension are injected into a blastocyst immobilised by a holding pipette in a micromanipulation apparatus. Then the embryos are incubated for at least 3 hours, possibly overnight.
In a preferred embodiment of the invention, genetically manipulated totipotent cells are used in order to obtain transgenic animals.
There are no restrictions regarding the type of genetic alteration of the totipotent cells; genes may be overexpressed, mutated or, in order to produce so-called knock-out animals, switched off; furthermore, foreign genes may be inserted or intrachromosomal deletions may be carried out. The genetic modification may be carried out on one or both alleles; this latter approach has been described for example by Hilberg and Wagner, 1992, for switching off the c-jun gene. The fact that the present invention allows genetic modification on both alleles is particularly advantageous; with the methods of the prior art it was only possible to obtain transgenic animals in which both alleles had the desired modification, after further crossing and tedious breeding of animals which had a genetic modification on one allele.
The genetic manipulation of the totipotent cells may be carried out by conventional methods. Generally, plasmids are used, preferably linearised plasmids, which carry the desired genetic modification. With a view to the selectability of the genetically modified ES cells, the plasmids preferably contain a marker gene, e.g. the neomycin, hygromycin or puromycin resistance gene, under the control of a promoter. With a view to the expression of a gene contained on the plasmid in the host cells the plasmid may contain gene expression control sequences, e.g. a strong promoter which is functional in ES cells or EG cells, such as the PGK (phosphoglycerol kinase) promoter.
The methods by which the plasmid is introduced into the cells are standard methods known from the literature for in vitro transfer of DNA into mammalian cells, such as electroporation; calcium phosphate precipitation or methods based on receptor-mediated endocytosis, disclosed in WO 93/07283, for example.
Another method of introducing genetic changes in the totipotent cells makes use of viruses, e.g. recombinant retroviral vectors; with regard to sequence sections contained on the vector which allow the selection of genetically modified cells or expression in the cell, basically the same applies as has already been stated with regard to the plasmids (Wagner and Keller, 1992; Stewart et al., 1985).
Using the process according to the invention it is routinely possible to produce viable and fertile transgenic mammals, particularly ES or EG mice, from totipotent cells genetically modified in vitro.
Using the process according to the invention, transgenic animals can be reproducibly created, inter alia from genetically manipulated totipotent cells which overexpress a specific gene, for example, or in which a specific gene has been inactivated, and these transgenic animals may be used, inter alia, for studies of gene function or for the production of proteins. Compared with conventional methods of producing transgenic animals, the process according to the invention provides an effective, rapid and economical method of producing mutant animal foetuses, particularly mouse foetuses, as well as transgenic strains directly from totipotent cells in which the desired genetic modifications have been made.
All three ES cell lines designated D3, R1 and GS1 investigated for the purposes of the present invention formed germline chimeras after injection into diploid blastocysts. When they were injected into tetraploid blastocysts, live ES mice were obtained from R1 and GS1 cells. With D3 cells it was not possible to produce live ES mice, even with cells from an early passage (passage 9), after injection of ES cells into tetraploid blastocysts. This accords with previous observations from aggregation experiments (Nagy et al., 1990; see also Table 2) with these cells. The inability of D3 cells to form viable ES mice presumably cannot be put down to the fact that these cells have lost their development potential; D3 cells have frequently been used in so-called gene targeting experiments in which, after they have been injected into diploid blastocysts, a high rate of chimerism and germline chimeras have been obtained (Urbxc3xa1nek et al., 1994; Wang et al., 1992; Wang et al., 1994; cf. also Tab.1). However, it is possible that the potential of D3 cells to differentiate into a few cell types which are critical for adapting the foetus to post-natal life is affected by unknown genetic or epigenetic changes. This assumption is supported by the observation that D3 cells are capable of producing foetuses which develop up to the normal birth date but the newborn are incapable of maintaining breathing, and they have a high birth weight and polydactyly and die at birth. These characteristics remind one of the phenotypical features of mice which lack the imprinted Igf2/Mpr gene (Wang et al., 1994; Lau et al., 1994); it might therefore be the case that imprinted genes or genes which regulate the growth of the foetuses are responsible for the effect observed. Whereas in the environment of host cells of diploid embryos defective functions of the ES cells might be complemented by the host cells, the development potential of being able to form all functional cell types, which is inherent in the D3 cells because of their lack of differentiation, would appear to be restricted in an environment derived totally from ES cells. The introduction of different wild-type ES cells into tetraploid embryos, conveniently in series of tests, may therefore be used as a fast and reliable test for checking the suitability of ES cells for use within the scope of the present invention.
The genetic background of the mouse strains from which the various ES cells originate could be another factor which influences the viability of the ES mice. All the ES cell lines used within the scope of the present invention originated from mouse strain 129:the R1 cells originated from a mouse blastocyst from a cross between the sub-strains 129/Sv and 129/Sv-CP (Nagy et al., 1993); GS1 cells originated from 129/Sv/Ev. D3-cells (Doetchmann et al., 1985) and J1 cells (Li et al., 1992) originated from 129/Sv or 129/terSv. TT2 cells which also yielded ES mice originated from an F1 hybrid strain (C57BL/6xc3x97CBA) (Yagi et al., 1993). On the basis of the results obtained within the scope of the present invention as well as earlier studies (Nagy et al., 1993, Ueda et al., 1995) we cannot rule out the possibility that ES cell lines derived from different strains or sub-strains of mice have different capacities to form viable ES mice.
The efficiency in the production of newborn ES mice by injection of wild-type R1 cells into tetraploid embryos (14%) was greater than the production by aggregation (6% within the scope of the present invention or 7% in the study described by Nagy et al., 1993). These results are in agreement with a comparison between the aggregation method and the method by injecting ES cells into diploid embryos (Wood et al., 1993). The use of tetraploid blastocysts according to the invention for the injection method showed that some selected R1 cell clones which had been cultivated in vitro for longer than 24 passages (e.g. R169.2.3 and R-fra3), still had the ability to produce viable ES mice. These findings are remarkable, particularly in view of the results of earlier aggregation experiments in which wild-type R1 cells lost their ability to produce viable ES mice after passage 14 (Nagy et al., 1993). The reasons why the injection of ES cells into tetraploid blastocysts reproducibly leads to the formation of ES mice are not totally clear. Since ES cells are obtained originally from ICM of blastocysts and are also very similar to these ICM cells (Beddington and Robertson, 1989), it is conceivable that both the spatial proximity of ES cells and ICM and their compatibility in development are responsible for this effect. This assumption is further supported by the observation, comparison tests, that the efficiency of producing chimeric mice was lower when ES cells were introduced into diploid morulae below the xe2x80x9czona pellucidaxe2x80x9d than when the conventional blastocyst injection method was used (injection into diploid blastocysts).
The high efficiency of the method according to the invention makes it superior to the methods of the prior art (aggregation of ES cells with tetraploid blastocysts or injection into diploid blastocysts) and offers the only possible method at present of creating mutant mice directly from genetically modified totipotent cells.
The production of viable mutant mice directly from genetically manipulated totipotent cells has numerous advantages. Since the foetal tissues are derived totally from totipotent cells which can be genetically modified, this technique provides a direct method of producing foetal material of pure ES or EG cell origin for cell-biological, molecular-biological or genetic studies (Forrester et al., 1991; Carmeliet et al., 1996).
ES foetuses reproduce the expression patterns of specific genes, such as for example the Pax5 gene or a xe2x80x9ctrappedxe2x80x9d gene, in a reliable manner, compared with foetuses produced by crossing heterozygotic mutant mice obtained from the same ES cells. Advantageously, ES foetuses can be used for expression studies, since they allow rapid production of foetal material (a few days) whereas conventional breeding normally takes four to five months. In addition, the reliable and reproducible expression pattern in ES foetuses minimises any possible complications in conventional chimeric tissues which by definition consist of both wild-type and mutated ES cells. Therefore, this technique is useful for studying gene function or for identifying new genes, e.g. in xe2x80x9cgene-trapxe2x80x9d studies (Skarnes, 1993). It has been shown that, with the aid of the method according to the invention (injection of ES cells into tetraploid blastocysts), mutant mouse lines, e.g. c-fos transgenes and fra-1 xe2x80x9cknock-outxe2x80x9d mice can be produced directly from mutant ES cells in an efficient manner. The process according to the invention makes it possible to produce transgenic mouse lines from ES cells or EG cells which had been preselected for the integration and expression of transgenes. The efficiency of producing mice which overexpress a specific gene is thereby improved significantly, compared with the conventional injection method in which diploid blastocysts are used. The process according to the invention has been used in a number of studies into gene overexpression and inactivation.
Furthermore, using this method, it is possible to produce mutant tissue for studying specific effects if inactivation or overexpression should lead to death or impaired gametogenesis in heterozygotic mutants or even in chimeras (see for example Carmeliet et al., 1996).
Finally, the process according to the invention offers the possibility of producing mutant animal strains, particularly breeds of mice, rapidly and economically and of having quick access to mutant foetuses and animals, which is a major advantage for research in the field of mice genetics.
As well as the production of transgenic animals, the process according to the invention may also be used to produce non-genetically modified ES animals or EG animals which have specific desirable qualities. For this, totipotent cells are used which are preselected for the desired qualities by culture experiments, in order to obtain identical animals with the required qualities.